The subject of the present invention is a new process for the preparation of CMP-activated (cytidine-5'-monophosphate), fluorescing or absorbing as well as new CMP-activated fluorescing or absorbing sialic acids.
Sialic acids (also called acylneuraminic acids) are components of many bacterial and animal glycoproteins and glycolipids. In the case of inflammations, tissue-destroying processes and certain tumour diseases, higher concentrations of sialic acids, especially of 5-N-acetylneuraminic acid, and increased activities of so-called sialyl transferases are also found in the blood plasma. The latter incorporate in a physiological manner the sialic acid intracellularly into glycoconjugates; it is a question of glycoprotein enzymes which have a definite specificity for the glycan sequence of the glycoconjugate acceptor into which the sialic acid is incorporated and for the glycosidic binding type in which the incorporated sialic acid is chemically bound. Not only the detection of the sialic acid concentration in the blood and tissues, especially also the sialylation pattern on the cell membrane surface, but also the detection of the sialyl transferase activity and specificity in cells or body fluids is, to an increasing extent, interesting for medical diagnosis.
It is problematical in the case of the detection of such enzymes that they are normally present in extraordinarily low activity so that direct detection methods are difficult. Therefore, according to the state of the art, radioactively-labelled CMP-activated sialic acids are used as substrate in order to determine sialyl transferase-catalysed reactions (cf. Beyer et al., Adv. Enzymol. Relat. Areas Mol. Biol., 52, 23-175 (1981)). Apart from the fact that, in many places, it is difficult to handle radioactive substances, these processes are time-consuming and expensive.
Therefore, there is a need for a simpler labelling substance which is problem-free to handle with which also small amounts of reaction products of the sialyl transferase reaction can be detected certainly, simply and sensitively.
Therefore, in Eur. J. Biochem., 177,583-589 (1988), a process is described of coupling 9-amino-5-N-acetyl-neuraminic acid with fluoresceinyl isothiocyanate which, according to the process described by Gross and Brossmer, Glycoconjugate J., (1987), Volume 4, 145-176, and Eur. J. Biochem., (1987) 168, 595-602, can be coupled with CMP. The fluorescein-labelled CMP-sialic acid prepared in this way proves, as indicator, to be superior to the known radioactively-labelled CMP-sialic acids. Not only the preparation of the fluoresceinyl-N-acetyl-neuraminic acid but also the subsequent CMP activation proceed, on the basis of the poor reaction and the numerous purification steps, only with a yield of about 5%, referred to the initially used 9-amino-5-N-acetyl-neuraminic acid. Therefore, there is a need to prepare this substance in a simpler way and with higher yields and thus to make it available more economically.